Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7 −/− mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.Staphylococcus aureus | α-toxin | adherens junctions | MRSA | PLEKHA7

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.HCV IRES | translation initiation | human ribosomes | single-molecule FRET P rotein synthesis is a central process in health and disease (1, 2). The basic steps in translation have been mapped by genetic, biochemical, structural, and mechanistic studies. However, how translation is regulated and subverted, for example, during viral infection, remains poorly understood, especially in eukaryotes. All viruses compete for the cellular translation machinery to synthesize viral proteins required for virus proliferation. To that end, many viruses contain a structured internal ribosome entry site (IRES) in the 5′ untranslated region of their genome, which allows them to bypass the requirement for certain translation initiation factors. How IRESs achieve this goal remains unclear.Recently, it has been shown that structurally and evolutionarily very diverse IRESs, such as hepatitis C virus (HCV) IRES, cricket paralysis virus (CrPV) IRES (3), and others (4), require ribosomal protein eS25 (RPS25) for efficient translation initiation, indicating that RPS25/IRES interactions could be a universal feature of IRES-mediated translation. RPS25 is located on the back of the head of the 40S ribosomal subunit, distal to the mRNA entry channel but proximal to different IRES RNAs as shown by cryo-EM structures of 80S:CrPV IRES and 80S:HCV IRES complexes. RPS25 is not essential for cap-dependent translation, suggesting that IRES/RPS25 interactions are required to bypass the requirement for the full set of initiation factors (3).Translation initiation is a multistep process, with kinetics and dynamic interrogation refractory to conventional biochemical and biophysical methods. Single-molecule approaches provide insight into compositional and conformational dynamics of these asynchronous processes by following individual molecular events in real time. In bacteria, single-molecule methods allow direct obser...

Highlights d The a-toxin receptor ADAM10 is clustered at junctions by the PLEKHA7-PDZD11 complex d Tspan33 docks ADAM10 to junctions by binding to the WW domain of PLEKHA7 d ADAM10 junctional clustering promotes the efficient formation of stable toxin pores d Loss of PLEKHA7-PDZD11 leads to toxin pore removal by endocytosis and cell survival

SUMMARY The innate immune system tightly regulates activation of interferon-stimulated genes (ISGs) to avoid inappropriate expression. Pathological ISG activation resulting from aberrant nucleic acid metabolism has been implicated in autoimmune disease, however the mechanisms governing ISG suppression are unknown. Through a genome-wide genetic screen, we identified DEAD-box helicase 6 (DDX6) as a suppressor of ISGs. Genetic ablation of DDX6 induced global upregulation of ISGs and other immune genes. ISG upregulation proved cell intrinsic, imposing an antiviral state and making cells refractory to divergent families of RNA viruses. Epistatic analysis revealed that ISG activation could not be overcome by deletion of canonical RNA sensors. However, DDX6 deficiency was suppressed by disrupting LSM1, a core component of mRNA degradation machinery, suggesting that dysregulation of RNA processing underlies ISG activation in DDX6 mutant. DDX6 is distinct among DExD/H helicases that regulate the antiviral response in its singular ability to negatively regulate immunity.

Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.

Staphylococcus aureus is both a major bacterial pathogen as well as a common member of the human skin microbiota. Due to its widespread prevalence as an asymptomatic skin colonizer and its importance as a source of skin and soft tissue infections, an improved understanding of how S. aureus attaches to, grows within, and breaches the stratified layers of the epidermis is of critical importance. Three-dimensional organotypic human skin culture models are informative and tractable experimental systems for future investigations of the interactions between S. aureus and the multi-faceted skin tissue. We propose that S. aureus virulence factors, primarily appreciated for their role in pathogenesis of invasive infections, play alternative roles in promoting asymptomatic bacterial growth within the skin. Experimental manipulations of these cultures will provide insight into the many poorly understood molecular interactions occurring at the interface between S. aureus and stratified human skin tissue.

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.

Highlights d Staphylococcus aureus skin infection leads to specific and functional IgE antibodies d Skin-initiated immunity protects against secondary lung and skin infections d Mast cells and IgE antibodies play key roles in acquired resistance to S. aureus d IgE-activated mast cells interfere with S. aureus growth and toxicity